Analytical package and method



C. J. SALIVAR ETAL 3,446,596

ANALYTICAL PACKAGE AND METHOD May 27, 1969 Filed April 2, 1965 United States Patent US. Cl. 23-230 8 Claims ABSTRACT OF THE DISCLOSURE A device for detecting specified substances in liquid materials, such as the detection of nitrites in urine, comprises a semi-rigid outer receptacle within which is a tightly closed frangible container for a reagent solution such as u-naph'thylamine and sulfanilic acid stabilized with a reducing substance, such as sodium sulfite. One end of the semi-rigid receptacle is closed with an absorbent plug, for example cotton. In use, the end of the receptacle containing the absorbent plug is moistened with the liquid material to be tested, and the semi-rigid outer receptacle is squeezed to break the inner frangible container and release the reagents. The presence of a specified substance is indicated by the development of color in the absorbent plug.

This invention relates to analytical packages and methods of utilizing such packages in the detection of substances by means of chromogenic chemical reactions.

Briefly, the invention is directed to an analytical package containing a reagent solution capable of forming a colored reaction product in the presence of a specified substance, the solution being packaged in such a way as to preserve the reagent against deterioration prior to use, to permit ready liberation of the reagent solution into a reaction zone at the desired time, and to provide a convenient reaction matrix, wherein the reagent solution and a solution suspected of containing the specified substance may be readily contacted when desired. The invention also includes methods of using the' packages of the invention in the examination of solutions suspected of containing such specified substances. The invention further includes novel stabilized reagent compositions for use in the detection of nitrites.

Among the objects of the invention may be mentioned the provision of analytical packages adapted for use in the detection in solutions of substances of biochemical interest; the provision of packages of the type mentioned which utilize chromogenic chemical reactions; the provision of such packages which are easy and convenient to use; the provision of packages of this character which may be successfully stored for extended periods without substantial deterioration; the provision of methods of using analytical packages of the type mentioned; and the provision of stabilized reagent compositions for use in the detection of nitrites. Other objects and features will be in part apparent and in part pointed out hereinafter.

The invention accordingly comprises the products and methods hereinafter described, the scope of the invention being indicated in the following claims.

In the accompanying drawings in which one of various possible embodiments of the invention is illustrated:

FIG. 1 is a vertical cross-sectional view of the package 3,446,596 Patented May 27, 1969 of the invention illustrating one embodiment of the invention, .and

FIG. 2 is a section taken on line 22 of FIG. 1.

The present invention is directed to novel analytical packages adapted to the ready and convenient detection of traces of substances of chemical interest in solution. A solution of a chemical reagent or reagents capable of generating a colored reaction product in the presence of a specified substance is tightly enclosed or sealed in an inner container of the package from which the solution may readily be liberated when desired. The inner container is enclosed within an outer casing, receptacle or container which affords protection for the inner container prior to use but which is adapted to be squeezed to effect rupture of the inner container. The outer container may be a tube, for example, closed at one end and having a wad or plug of porous, preferably absorbent, material in its other end. The wad or plug of porous material is preferably disposed at one end of the tube and functions as a porous stopper, but may be disposed inward of one end of the tube if desired. Optionally, the end having the porous material disposed therein may be covered with a protective cap to exclude dust and other contaminants.

Referring now to the drawings, FIG. 1 shows a package of the invention generally designated 1 as comprising an outer container 3 and an inner container 5 enclosed within the outer container. As shown, outer container 3 is a tube composed of semi-rigid material and inner container 5 is a sealed capsule or ampul of frangible material. At 7 is indicated a reagent solution enclosed within inner container 5. One end of outer container 3 is closed with a stopper 9 which may be removed in assembling the package 1. It will be understood that in place of stopper 9 the end of outer container 3 may be closed by having the end integral with the walls of the container. At the other end'of outer container 3 is a plug 11 of porous material which acts as a stopper for container 3 and as a reaction matrix for the chromogenic reaction. At 13 is an optional closure which may, if desired, be used to cover and, protect plug 11.

While the inner container 5 for the reagent solution is preferably a frangible capsule, as shown in FIG. 1, it will be understood that other types of frangible containers may be employed. The important characteristics of the inner container are (1) that it be tightly closed or sealed so as to prevent evaporation or leakage of the reagent solution and to protect the solution from exposure to air; and (2) that it be readily rupturable or puncturable so as'to permit ready liberation of the reagent solution into the outer container when a test is to be performed. Frangible containers which may be used in the practice of the invention include, for example, thin-walled glass capsules and a stoppered semi-rigid plastic tube designed to permit stopper to pop out when pressure is applied to the walls of the tube as by squeezing the outer container.

Outer container 3 is preferably fabricated of a clear, semi-rigid material, such as celluloid, cellulose acetate butyrate or polyethylene, for example, so that the frangible inner container may be readily ruptured or punctured by the application of moderate pressure to the walls of the outer container. A colorless, transparent material is generally advantageous because it permits ready observation of any color changes taking place Within the area of the reaction matrix.

The plug 11 of porous material is preferably an inert absorbent material, such as cotton, for example, which will absorb and retain a significant volume of the test liquid or solution. Other suitable porous materials include rayon, nylon, the polyacrylonitrile synthetic fiber sold under the trademark Orlon, and similar fibers.

In the practice of the invention, it is preferred and customary to enclose within inner container 5 the reagent solution containing a dissolved reagent or reagents capable of reacting with a substance whose detection is desired, to form a colored reaction product. However, in the case of certain reagents such as Fehlings solution, for example, which consist of two components and which must be freshly prepared shortly before use, it is preferable to separate the components and yet permit them to be readily intermixed or contacted when desired. For this purpose, the separate components of the reagent may be separately enclosed within sealed frangible containers of the type previously described and both containers then enclosed within an outer container as before. Upon squeezing the Walls of the outer container, the reagent solutions in both containers are released into the outer container and become intermixed. Alternatively, one of the components of the reagent may be enclosed within a sealed container and the other component of the reagent may be absorbed by the plug of porous material and remain thereon in a dried condition. In this embodiment, the single sealed container is ruptured as previously described and the solution of the one reagent component released then contacts the porous material and becomes intermixed with the other reagent component carried thereby so that the resulting mixed reagent is capable of generating a colored reaction product in the presence of a specified substance.

Where there is a possibility that the substance to be detected may be absorbed by the porous material from the atmosphere and/ or where one of the components of the reagent employed is carried by the porous material, it is important to protect the porous material from contamination. This may be done, for example, by hermetically sealing the package of the invention within a third container or by covering the end of the outer container in which the porous material is disposed with a tight fitting closure or cap.

Some chromogenic analytical reactions are preferably carried out at elevated temperatures. In such instances the analytical package may be preheated, in an oven for example, for a sufficient time to warm the reagent solution to the desired temperature prior to carrying out the test.

The analytical packages of the invention may be utilized in the detection of a number of substances for analytical or diagnostic purposes. For example, the detection of traces of nitrites in various environments is sometimes of importance. Many years ago P. Griess disclosed that u-naphthalyarnine and sulfanilic acid react in the presence of nitrite to form an intensely red reaction product. The application of this color reaction in microbiology and in testing the purity of water supplies is known. Also, it has been introduced into the field of clinical chemistry in the diagnosis of urinary infections. Many organisms causing urinary tract infections produce nitrite from nitrate which is usually present in the urine. Detection of ntirite in the urine, by the Griess reaction, for example, indicates the presence of urinary tract infection due to nitrate-reducing organisms.

The ordinary Griess reagent, consisting of an acid solution of sulfanilic acid and a-naphthylamine, is known to be rather unstable. The freshly prepared solution is colorless, but on storage it is degraded by light and oxygen, turning pink and eventually depositing a precipitate.

In accordance with the present invention, it has further been found that the inclusion of traces of certain reducing agents in the Griess reagent solution produces a stabilized solution which has an extended shelf life when stored in a sealed container. The preferred stabilizers are soluble sulfite salts, such as sodium sulfite, sodium bisulfite, and potassium metabisulfite. Also effective, although to a lesser extent, are sulfurous acid, metallic tin (granules) and thioglycolic acid. Between approximately 0.01-1%, preferably 0.1%, weight by volume in solution of one of the above stabilizers is sufficient to produce a stabilizing effect.

The following examples further illustrate the invention.

EXAMPLE 1 A stabilized Griess reagent was prepared according to the following formula:

Component: Amount a-Naphthylamine hydrochloride gm 6.75 Sulfanilic acid gm 11.25 Glacial acetic acid ml 300 Sodium sulfite gm 3 Distilled water, qs. 3000 ml.

EXAMPLE 2 The package described in Example 1 was used to detect nitrite in the following manner. The dust cap was removed, and the end of the tube containing the cotton plug was dipped into a solution, such as urine, suspected of containing nitrite. The walls of the plastic tube were then squeezed sufficient to break the glass capsule, thereby releasing the Griess reagent to react with the test solution held by the cotton plug. A pink color developed rapidly in the presence of traces of nitrite. As little as 1 microgram of nitrite/ml. solution may be detected. Semiquantitative estimation of nitrite content may be made by comparison with standards.

EXAMPLE 3 A stabilized Greiss' reagent was prepared according to the following formula.

Component: Amount u-Naphthylamine hydrochloride gm 6.75 Sulfanilic acid gm 11.25 Glacial acetic acid ml 300 Sodium bisulfite gm 3 Distilled water, qs., 3000 ml.

This reagent was stable toward discoloration or precipitation for long periods.

This reagent was packaged as described in Example 1 and used as described in Example 2 with comparable results.

It is well known, of course, that the formation of dyestuffs by the diazotization of an aromatic amine with nitrite (nitrous acid) followed by the coupling of the resulting diazo compound with a phenol or aromatic amine is a general reaction which may be accomplished with many systems other than the a-naphthylamine-sulfanilic acid combination. Many such other chromogenic systems are adaptable to use in the present invention.

While the novel packages, methods and reagent compositions of the invention have been particularly described with respect to the detection of nitrite using a modification of the Griess test, they are not limited thereto. For example, the following table lists other reagents adapted to incorporation into the novel packages of the invention and the corresponding substances whose detection may be accomplished by the novel methods disclosed herein.

TABLE 1 Substance to be Reagent detected (1) Bened)icts solution (alkaline cupric citrate Reducing sugars.

solution (2) Fehlings solution (alkaline cupric tartrate D0.

solution).

(6) Aluminon solution Aluminum. (7) Bial's reagent (orcinol-ferric chloride solution) Pentoses. (8) o-Tolidine solution Chlorine.

In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

As many changes could be made in the above products and methods without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

What is claimed is:

1. The method of detecting the presence of nitrites in liquids by means of a chromogenic chemical reaction utilizing a package comprising an outer receptacle constituted by a tube closed at one end and having a plug of absorbent material in its other end adapted to be contacted by a test substance entering the tube through said other end, a tightly-closed frangible container enclosed within said tube and containing a reagent solution comprising atnaphthylamine and sulfanilic acid stabilized with a small amount of a reducing substance, said tube being formed of semi-rigid material so as normally to protect said frangible container but being adapted to be squeezed so as to eifect rupture of said container thereby to release said reagent solution therefrom to contact said absorbent material, comprising the steps of moistening the absorbent material in said tube with a solution suspected of containing a nitrite, squeezing the walls of said tube to rupture said frangible container and release said reagent solution thereby contacting the reagent solution with the moistened absorbent material, and examining the absorbent material for the presence of a colored reaction product.

2. The method according to claim 1 wherein the solution contains between approximately 0.01 and 1% weight by volume in solution of a soluble sulfite as a stabilizer.

3. An analytical package for use in the detection of nitrites in liquids by means of a chromogenic chemical reaction, the said package comprising an outer receptacle constituted by a tube closed at one end and having a plug of absorbent material in its other end adapted to be contacted by a nitrite test substance entering the tube through said other end, a sealed frangible capsule enclosed within said tube containing an aqueous solution of cz-naphthylamine and sulfanilic acid stabilized with a small amount of a reducing substance, said tube being formed of transparent, semi-rigid material so as normally to protect said capsule but being adapted to be squeezed so as to effect rupture of said capsule thereby to release said aqueous solution therefrom to contact said cotton plug.

4. An analytical package as set forth in claim 3 wherein said other end of said tube is covered by a removable closure to protect said plug from contamination.

5. An analytical package as set forth in claim 3 wherein said aqueous solution contains between approximately 0.01 and 1% weight by volume in solution of a soluble sulfite as a stabilizer.

6. A reagent composition for use in the detection of nitrites comprising an acid solution containing a-naphthylamine, sulfanilic acid and between approximately 0.01 and 1% weight by volume in solution of a stabilizer selected from the group consisting of water soluble sulfite salts, sulfurous acid, metallic tin and thioglycolic acid.

7. A reagent composition for use in the detection of nitrites comprising an acid solution containing u-naphthylamine, sulfanilic acid and between approximately 0.01 and 1% weight by volume in solution of a water soluble sulfite salt.

8. A reagent composition for use in the detection of nitrites comprising an acid solution containing a-naphthylamine, sulfanilic acid and between approximately 0.01

and 1% weight by volume in solution of sodium sulfite.

References Cited UNITED STATES PATENTS 3,033,655 5/1962 Grosskopf 23254 OTHER REFERENCES Rider, B. F., with Mellon, M. G.: Analytical Chemistry, 18, pp. 96-99' (1946).

MORRIS O. WOLK, Primary Examiner. E. A. KATZ, Assistant Examiner.

US. Cl. X.R. 23253; 252-408 

